In order to replicate, a retrovirus such as HIV must integrate a cDNA copy of its RNA genome into a chromosome of the host. Although the cDNA integration system is a promising target for antiretroviral therapy, no clinically useful inhibitors have yet been developed. Our proposed studies are designed to provide mechanistic information useful in developing such inhibitors. Integration in vivo is carried out by a sub-viral particle containing the viral cDNA complexed with proteins. Such "preintegration complexes" (PICs) can be isolated from infected cells and when presented with a target DNA in vitro can carry out integration. We have been engaged in a long term study of the composition, organization and activities of PICs. We propose to continue this work in the renewal and investigate the implications with studies of viral replication in cell culture. We propose three specific aims 1) we will further improve methods for the large-scale production of PICs and their biochemical analysis, particularly the detection and quantitation of complex components using LC-MS and other methods. 2) We will investigate the organization of PICs using chromatin immunoprecipitation (ChIP) assays, EM, and activity based tests. 3) We will investigate the function of PIC components using cells and viruses containing appropriate mutations. Virus growth will be monitored using quantitative PCR assays, including a new method we devised for quantitating integration in vivo. Highlight of our studies include the findings of three cellular proteins associated with PICs, the Ku heterodimer and the architectural DNA binding protein HMG I (Y). The discovery of Ku protein in PICs together with other work has lead to a new model for the role of the host cell non-homologous DNA end joining pathway in retroviral growth. Long term, these studies, together with data on protein structures, will provide a high-resolution picture of the organization and function of preintegration complexes.